Mango (Mangifera indica L.) is one of the most popular and widely cultivated tropical fruit, mainly due to its attractive flavour. Based on findings (Mukherjee, 1997 and Bompard and Schnell, 1997), the center of origin and diversity of the genus Mangifera is now firmly established in Southeast Asia. Several mango cultivars are grown throughout the world and are known to differ evidently in their flavour characteristics. These attributes have resulted in an increased significance of the commercial value of mango.
Volatiles form an indispensable component of flavour in mangoes therefore extensive labor and effort has been invested to decipher this volatile composition of various mango cultivars (Pino and Mesa 2006; Macleod and Pieris 1984; Pandit et al 2009a). Subsequently, all these efforts have put forth together a vast diversity of volatile compounds portrayed by mango cultivars. More than 300 volatile compounds have been identified as free forms and approximately 70 compounds as glycosidically bound compounds. This variability in volatile compounds has been established to depend on the cultivar, maturity stage of the fruit, part of the fruit and processing (Pino et al, 2005).
Amongst the diverse set of cultivars, Alphonso is a highly valued Indian mango cultivar and is grown mainly in the western part of India including Sindhudurg, Ratnagiri and Raigad districts and in the Konkan region of India. Its volatile composition relating to various stages of development and ripening have been analyzed by various researchers (Pandit et al 2009). Although studies revealed that the volatile profile of Alphonso mangoes is dominated by terpene hydrocarbons throughout fruit development and ripening, their impact on the overall flavour of ripe Alphonso was found to be relatively less owing to their high odour detection threshold. Further, to support the theory of minimal impact of terpene hydrocarbons on the flavour profile of Alphonso, studies by Pandit et al. (2009b), and Kulkarni et al (2012) have indicated that similar pattern of terpenes is also detected in flowers and leaves of Alphonso mango and it is the lactones and furanones which are particularly synthesized during the late ripening stages that have significantly low odour detection threshold and thus contribute majority of flavour imparting sweet fruity caramel like notes to ripe Alphonso fruits.
Furanones are widely distributed in nature and have a very low odour threshold value in water i.e. 4×10−5 mg/kg; hence its effect on food aroma is considerable (Latrasse, 1991). In Alphonso mango, furanones comprise 4-hydroxy-2,5-dimethyl-3(2H)-furanone (furaneol) and its methyl ether, 2,5-dimethyl-4-methoxy-3(2H)-furanone (mesifuran). The importance of these compounds to the flavour of Alphonso mangoes was first evaluated organoleptically by Wilson III et al (1996). Both these compounds have an odour detection threshold of 10 and 0.03 ppb, respectively and thus have high ‘aroma value’. Alphonso mangoes showed quantitative dominance of these compounds when compared with other cultivars (Pandit et al. 2009a).
Further, M. Sakho et al (1997) disclose carbohydrate and aglycon moieties released, respectively, by acid and enzymatic hydrolysis of African mango pulp extracts containing glycosidically bound compounds of which vanillin was identified to be one of the components. Vanillin is an industrially produced aroma compound which is a crystalline powder in its isolated form. Due to high consumer perception, naturally prepared vanillin is considered a more suitable food additive and hence it has higher market value as compared to its synthetic version (Priefert et al. 2001). For improved vanillin production by ferulic acid bio-conversion, genetically engineered strain cloned with genes from P. fluorescens has been developed by Barghini et al, which was capable of producing vanillin without accumulation of undesirable metabolites. Product inhibition is one of the several problems that the vanillin production encounters (Biotransformation of Waste Biomass into High Value Biochemicals, Satinder, Kaur et al).
Considering the lack of production methods for the synthesis of vanillin and furanones, importance is given to these volatile compounds due to their use in the food and fragrance industry, as chemical intermediates in the production of pharmaceuticals and other fine chemicals. High importance of furanones for the food industry has promoted standardization of their chemical synthesis by various researchers, however; the identification of a suitable biosynthetic process for the production of furaneol and mesifuran is a field still in infancy (Schwab 2013).
Research studies available in the art do not provide for synthesis of vanillin and mesifuran therefore limiting the use of O-methyltransferase. Levid et al (2002) partially purified and biochemically characterized S-adenosyl-L-methionine (SAM) dependent O-methyltransferase from Fragaria ananassa i.e. strawberry fruit extracts which displayed active conversion of furaneol to mesifuran in an assay reaction. This was later supported by the molecular isolation of corresponding cDNA followed by the expression of its recombinant protein (FaOMT) which demonstrated successful synthesis of mesifuran from furaneol in an in vitro assay reaction (Wein et al 2002).
Kulkarni et al (2013) have characterized an enone oxidoreductase (MiE0) which catalysed the synthesis of furaneol, a precursor molecule of mesifuran from Alphonso mango to study biosynthesis of furanones in mangoes. Further, ethylene treatment resulted in early and exceptionally enhanced synthesis of mesifuran in Alphonso mango (Chidley et al 2013).
However, in spite of the synthesis of vanillin and mesifuran using biotechnological applications, there is still a need in the art to synthesize these flavour imparting components in higher yield. Therefore the present inventors have isolated and expressed cDNA encoding O-methyltransferase (O-MTS) which catalyze the synthesis of mesifuran from furaneol and vanillin from protocatechuic aldehyde in an in vitro assay reaction.